Pan-inhibition of the three H2S synthesizing enzymes restrains tumor progression and immunosuppression in breast cancer

Background Hydrogen sulfide (H2S) is a significant endogenous mediator that has been implicated in the progression of various forms of cancer including breast cancer (BC). Cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST) are the three principal mammalian enzymes responsible for H2S production. Overexpression of CBS, CSE and 3MST was found to be associated with poor prognosis of BC patients. Moreover, H2S was linked to an immune-suppressive tumor microenvironment in BC. Recently it was observed that BC cells, in response to single or dual inhibition of H2S synthesizing enzymes, develop an escape mechanism by overexpressing alternative sources of H2S generation. Thus, the aim of this work is to escape the H2S compensatory mechanism by pan repressing the three enzymes using microRNAs (miRNAs) and to investigate their impact on the oncogenic and immunogenic profile of BC cells. Methods BC female patients (n = 25) were recruited. In-silico analysis was used to identify miRNAs targeting CBS, CSE, and 3MST. MDA-MB-231 cells were cultured and transfected using oligonucleotides. Total RNA was extracted using Biazol, reverse transcribed and quantified using qRT-PCR. H2S levels were measured using AzMc assay. BC hallmarks were assessed using trans-well migration, wound healing, MTT, and colony forming assays. Results miR-193a and miR-548c were validated by eight different bioinformatics software to simultaneously target CBS, CSE and 3MST. MiR-193a and miR-548c were significantly downregulated in BC tissues compared to their non-cancerous counterparts. Ectopic expression of miR-193a and miR-548c in MDA-MB-231 TNBC cells resulted in a marked repression of CBS, CSE, and 3MST transcript and protein levels, a significant decrease in H2S levels, reduction in cellular viability, inhibition of migration and colony forming ability, repression of immune-suppressor proteins GAL3 GAL9, and CD155 and upregulation of the immunostimulatory MICA and MICB proteins. Conclusion This study sheds the light onto miR-193a and miR-548c as potential pan-repressors of the H2S synthesizing enzymes. and identifies them as novel tumor suppressor and immunomodulatory miRNAs in TNBC. Supplementary Information The online version contains supplementary material available at 10.1186/s12935-024-03317-1.


Introduction
H 2 S plays a complex role in cancer.It has both pro-tumor and anti-tumor effects depending on the concentration of H 2 S, the source of H 2 S (exogenous vs. endogenous) and the cancer model used [1][2][3][4].H 2 S -especially at lower concentrations and when it is produced by endogenous sources in cancer cells -can augment cancer progression by stimulating cancer cell growth, facilitating angiogenesis, and promoting resistance to chemotherapy [5,6].On the other hand, H 2 S -especially when applied exogenously, in the form of various H 2 S donor compounds -can also exert anti-tumor effects through induction of apoptosis and inhibition of cancer cell proliferation by reducing DNA synthesis and arresting the cell cycle [7,8].
Several studies have demonstrated that overproduction of H 2 S occurs in breast cancer (BC) and correlated the upregulation of H 2 S synthesizing-enzymes, namely cystathionine-β-synthase (CBS) and cystathionine-γlyase (CSE), with poor clinical prognosis [9].Wang et al., has shown that decreasing H 2 S level through CSE inhibition has led to inactivation of the JAK/STAT pathway via upregulation of SIRT1 [10].You et al. have demonstrated a feedback regulation cycle between CSE and STAT3 and proposed a direct involvement of STAT3 in the amelioration of CSE transcription and H 2 S production [11].CSE upregulation phosphorylates and activates STAT3 leading to augmentation of its own transcription in a positive-feedback manner [11].CSE upregulation phosphorylates and activates STAT3 leading to augmentation of its own transcription in a positive-feedback manner.
One of the substantial physiological processes for H 2 S is the involvement in immunosurveillance.In recent years, extensive research has been carried out to determine the role of H 2 S in immunomodulation and in tumor immune microenvironment (TIME) [12].Regulators of H 2 S-synthesizing machinery and H 2 S intracellular levels have been reported to modulate the TIME [13][14][15].In a study conducted by our group, where BC cells were co-cultured with natural killer (NK) cells, the decrease of either CBS-derived or CSE-derived H 2 S has led to an increase in NK cell-mediated catalytic activity [15].Moreover, CBS-derived H 2 S has exerted immunosuppressive activity by protecting BC cells from activated macrophage-generated ROS in macrophage-BC cell cocultures [14].As such, modulating H 2 S levels could also affect TIME.Dual inhibition of CBS and CSE in TNBC and HR + BC cells has affected the TIME by suppressing BC cells release of tumor necrosis factor alfa (TNFα), a cytokine that acts as an immune-suppressor within the TIME [13].Concomitantly, production of interferon gamma (IFN-γ) has restored the immune-stimulating conditions in the TIME [13].
3-Mercaptopyruvate sulfurtransferase (3MST), the third H 2 S-synthesizing enzyme, is well known for its critical physiological role in cellular metabolism and bioenergetics [16].In recent years, there has been growing interest in the role of 3MST in cancer progression [17].Some studies have shown that 3MST is upregulated in various types of cancers, including colon cancer [18,19], glioma [20], lung carcinoma [21], renal cancer [22], oral cancer [23], as well as in glioblastoma cell lines [24,25] but not adequately studied in BC.
The expression of various H 2 S biosynthetic enzymes can be directly controlled by miRNAs [26].The expression profiles of miRNAs that target H 2 S-synthesizing enzymes, have been found to be altered in different clinical oncological and non-oncological settings.MiR-203 has been found to regulate oxidative stress induced cell injury by regulating CBS expression and adjusting the levels of H 2 S production [27].In colorectal cancer, miR-559 was shown to target CBS thus reducing the accelerated cancer cell proliferation [28].On the contrary, few miRNAs have exhibited dual or multiple targeting ability for H 2 S-synhesizing enzymes, like miR-4317 that targets CBS and CSE together [29].Reports that describe miRNA targeting 3MST are not available.It was recently found by our research group that knocking down of CBS caused a compensatory increase in CSE expression rescuing H 2 S level [30].This inhibition-sensory behavior highlights compensatory mechanisms that maintain the level of H 2 S in BC cells.
Given the key role of 3MST in fostering cancer cell survival and augmenting cancer cell growth and proliferation [2,17,25,31], investigating its expression level in BC tissues could widen our understanding for the role of H 2 S synthesizing enzymes in BC tumorigenesis.Here, we screened in clinical breast cancer specimen the 3MST expression levels.Our data show that 3MST as well as CBS and CSE are significantly overexpressed in BC tissues compared to their non-cancerous counterparts.Furthermore, to escape the compensatory upregulation behavior of H 2 S-synthesizing enzymes, we performed insilico analysis to achieve pan-inhibition of the three H 2 S synthases simultaneously.In a "three birds, one stone" approach, we show that utilizing one miRNA that targets CBS, CSE, 3MST simultaneously could markedly and efficiently suppress BC hallmarks in TNBC cells and enhance the expression of immunomodulatory factors.

Clinical specimens
Breast tissues were collected from 25 BC female patients during conservative mastectomy or lumpectomy surgery at the National cancer Institute, Egypt.Tissues from both breast tumor and adjacent pathologically confirmed nontumor tissues from the safety margins (5-7 cm away from the tumor margin) were resected.All specimens were confirmed in the Department of Pathology, and relevant clinical data were collected.BC patients who had a previous history of BC or smoking or hypertension were eliminated.The specimens were snap frozen in liquid nitrogen and immediately stored at -80 °C.The Ethics Committee of the Faculty of Pharmacy and Biotechnology, the German University in Cairo ratified the study protocol in accordance with the ethical standards of the Declaration of Helsinki.All individuals signed informed written consent documents prior to their involvement in the study.

Cell culture and treatment
Culture of human TNBC cell line MDA-MB-231 was conducted in Dulbecco's modified Eagle's medium (DMEM) (Lonza, Switzerland) supplemented with 4.5 g/l glucose, 4 mmol/l L glutamine, 10% fetal bovine serum (Lonza, Germany) and 1% Penicillin-Streptomycin (Lonza, Germany) at 37 °C in 5% CO 2 atmosphere.Cells were passaged upon achieving 70-80% confluency.A stock solution of H 2 S donor (40 μm NaHS) was prepared using free DMEM.Co-treatment of the TNBC cells seeded in 96-well plates with the NaHS donor was performed for 24 h under normal growth conditions (37 °C in 5% CO 2 atmosphere) [33,34].Control cells in the H 2 S donor experiments were subjected to DMEM only.All cell experiments in this study were performed in triplicate and repeated at least three times [26].

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted from BC patients and cell line using Biazol (Invitrogen, USA) reagent.The integrity of RNA was verified by gel electrophoresis on 1% agarose.For gene expression assay, RNA was reversely transcribed by the High-Capacity cDNA Reverse Transcription Kit (4,368,814 -ThermoFisher Scientific, USA) while for miRNA expression quantification, RNA was reversely transcribed by the TaqMan™ Advanced miRNA cDNA Synthesis Kit (A28007 -ThermoFisher Scientific, USA) according to the company's instruction.RT-qPCR was performed in StepOne™ Plus (ABI, USA).All genes with their catalog number and assay ID are listed in Supplementary Table S1 in supplementary data.The housekeeping genes β-actin and 18s rRNA as well as miR-26b-5p were endogenous controls.The 2 −ΔΔCt method was applied to calculate relative expression [29,37].

Quantification of H 2 S production
H 2 S levels were measured using the H 2 S-sensitive fluorescent probe 7-azido-4-methylcoumarin (AzMC) (Sigma-Aldrich).TNBC cells were seeded in black 96-well plates with an optical bottom at 10,000 cells/well in 100 µl full DMEM and incubated at 37 °C and 5% CO 2 to allow cells to adhere.After 24 h seeding, the cells were transfected with the oligonucleotides of interest.Then, 48 h post-transfection, the supernatant was replaced by 100 µl of 100 µM of AzMC (Sigma-Aldrich) prepared in HBSS.After one hour of incubation at 37 °C in the dark, fluorescence was measured on the Wallac 1420 Victor reader with excitation and emission wavelengths of 355 and 460 nm, respectively.The final concentration of DMSO was kept constant at 0.1% in all conditions.Data analysis was performed after removal of the non-specific background fluorescence values [38].

Colony forming assay
Transfected cells were counted by hemocytometer and fostered in 6-well plates at 0.4 × 10 3 cells/well for 2 weeks.The formed cell colonies were fixed with 6% glutaraldehyde and stained with 0.2% crystal violet solution.Then, the stained cell colonies were manually counted [35].

Scratch test (wound healing assay)
Transfected cells at 90-95% confluence in 24-well plates were scraped by a sterile 10 µl micropipette tip vertically along a ruler.Then, cells were rinsed with PBS to remove detached cells and substituted with new low serum media (1% FBS).Afterwards, cells were observed for the migration rate at 0 and 24 h and wound closure was quantified with Zen2012 software (ZEISS Microscopy, Jena, Germany) by measuring the surface area of the scratch [40,41].

Transwell migration assay (Boyden chamber assay)
Transfected cells were seeded into the upper part of the 5-µm Transwell chamber (cellQART®, Germany) at concentration of 5 × 10 4 cells suspended in 300 µl of 1% full DMEM.The medium (700 µL) containing 10% FBS was added to the lower part.Through incubation under the conventional conditions, the non-migrating cells in the upper part were removed while the migrating cells in the lower part were fixed in 6% glutaraldehyde and stained with 0.2% crystal violet solution, followed by photography under an optical microscope.For accurate assessment, each insert was then transferred to an empty well containing 700 µl of the extraction solution (33% acetic acid) to lyse the cells, and 100 µl of each sample was transferred and measured at 595 nm using Wallac 1420 Victor reader.

Statistical analysis
Sample size was calculated by G*Power version 3.1.9.2; Germany with a power 80% and a level of significance (α) of 5% and expected effect size (1-β) of 0.8.Data are presented in the form of mean ± standard error of the mean (SEM).Student's t test was performed to compare between every two independent groups.Statistical significance was considered as p < 0.05.For multiple comparisons, one-way analysis of variance (One-way ANOVA) with post hoc analysis was used.Data were statistically analyzed using GraphPad Prism 8.00 software (GraphPad Software Inc., San Diego CA).

CBS, CSE and 3MST are overexpressed in BC tissues
A summary of the patients' characteristics is provided in Table 1.The average age of the BC patients was 46.36 years, with an age range of 26-72 years.According to molecular subtype, 52% of the patients were of luminal A subtype, 12% were luminal B, 28% were TNBC while only 8% were of HER2-enriched subtype.According to tumor grade, 8% of the patients had grade I BC, 64% had grade II while 28% had grade III.For lymphatic involvement, 60% of the patients had lymph node metastases.80% of the patients expressed high proliferative index Ki-67 levels.A small number of participants (2/25; 8%) were identified to have the invasive/infiltrating lobular carcinoma (ILC) histological subtype.Additionally, 64%, 56%, and 16% of the patients showed positive expression of ER, PR, and HER2, respectively.

In-silico analysis revealed miR-193a-3p and miR-548c-3p as the candidates of choice
The miRNA candidates selected to simultaneously target CBS, CSE, and 3MST were hsa-miR-193a-3p and has-miR-548c-3p.Their accession numbers and mature sequences were obtained (Supplementary Tables S2 and Table S4) and were introduced into the eight different computational algorithms.MiR-193a-3p was found to hit CBS 3'UTR sequence at 1 binding region, CSE CDS region at 2 different binding sites, and 3MST 3'UTR sequence at 1 binding region (Supplementary Table S3).MiR-548c-3p was found to hit CBS 3'UTR sequence at 6 different binding regions, CSE CDS at 9 different binding regions, and 3MST 3'UTR sequence at 1 binding region (Supplementary Table S5).

Discussion
BC remains the most prevalent tumor among women worldwide among all age categories [42].TNBC subtype of BC, which lacks ER, PR, and HER2 expression, is known as the most aggressive subtype of BC with a relatively high recurrence rate, especially within the first five years after diagnosis [43,44].It also has an increased tendency of metastasizing to other organs, including the liver, lungs, and brain [43].The standard treatment for TNBC includes surgery, radiation therapy, and chemotherapy which is usually given before the surgery as a neoadjuvant [45].However, TNBC is highly resistant to chemotherapy, making it more challenging to treat [46].Significant efforts are being focused on the development of targeted and immunotherapies that may improve the prognosis of TNBC patients [46].One such strategy involves using miRNAs [47].Due to the promising results shown by several preclinical studies using miRNAs as therapeutic targets for TNBC [48], clinical trials are underway to evaluate the effectiveness of clinical translation of miRNA-based therapy into practice for treatment of different cancers, including TNBC [49].
H 2 S, the most recently identified gasotransmitter member, has been found to be involved not only in the regulation of various physiological processes, but also in many pathophysiological events such as tumor progression [17].Dysregulation of H 2 S and its synthesizing enzymes have been linked to malignancies by either showing overexpression, or downregulation, highlighting that H 2 S and its synthesizing machinery have a tumor specific character [50,51].Our group has previously identified Fig. 3 Expression profiles of miR-193a-3p and miR-548c-3p in BC patients.MiR-193a-3p and miR-548c-3p expression profiles were analyzed in 25 BC patients using qRT-PCR and normalized to miR-26b-5p as an internal control.Screening of miR-193a-3p (A) and miR-548c-3p (B) in breast tissues showed a significant under expression compared to normal counterparts.;**= P < 0.01, **** = P < 0.0001 compared to noncancerous breast tissues the upregulation of CBS and CSE on the transcriptional level either in cell line or in clinical samples of different BC subtypes [29].These findings were supported by other studies that proved the involvement of H 2 S in many tumorigenic and immunosuppressive signaling pathways [52,53].Single or dual inhibition of CBS and CSE has significantly abrogated BC progression [29].However, our group has recently observed [54] that, at single targeting of CBS, CSE gets upregulated as a compensatory mechanism to save the diminishment of H 2 S production.In the light of these findings, this study aimed to investigate the expression level of the third H 2 S-synthesizing enzyme, 3MST, along with the other two enzymes, in BC patients.Additionally, this work intended to find miRNAs that simultaneously target CBS, CSE, and 3MST to avert the compensatory upregulation response by the untargeted enzyme(s).
Screening of 3MST in BC tissues showed an overexpression of this enzyme in all BC subtypes.Overexpression pattern of 3MST was also reported in tumor tissues of brain gliomas [20], colon cancer [19], lung carcinoma [21], oral squamous cell carcinoma [23], adenoid cystic carcinoma of the oral cavity [55], renal cell carcinomas [22], and bladder urothelial cell carcinoma [56,57], on the transcriptional level.This study was the first to demonstrate the association of 3MST with young age (< 40), pre-menopausal, large tumor size and high Ki BC patients.Lower 3MST expression levels were associated with larger tumor size in HCC [58].CBS was also reported to be downregulated in HCC patients' tissues [59].One study by Kaczor-Kamińska and colleagues have screened 3MST in mouse mammary gland cell line (NMuMG) and mouse mammary gland tumor cell line (4T1) [60].They found that relative gene expression of 3MST was higher in the tumor cell line when compared to the normal one.
In support of the in silico work, we noticed that ectopic expression of miR-193a-3p and miR-548c-3p in TNBC cell line caused a reduction in the transcript levels of CBS, CSE and 3MST.Subsequently, a significant impediment in the H 2 S production level was observed.On a similar note, previous results demonstrated the impact of miR-193a-3p on repressing ERK protein, a validated downstream target of H 2 S [65].Likewise, the impact of miR-548c-3p on HIF1-α, a signaling molecule for H 2 S, was reported by Du et al. [74].Nonetheless, our study was the first to unravel the impact of miR-193a-3p and miR-548c-3p on impeding H 2 S production in TNBC through tribunal targeting of CBS, CSE, and 3MST.
Over the past decades, intensive efforts have been directed to find novel cancer therapies that attenuate the immunosuppressive and enhance the immunostimulatory effects against malignancy [79].Indeed, immunomodulation is a promising approach in cancer therapy, including BC [80].GAL3, GAL9, and CD155 are examples of molecules that play an immunosuppressant role, impeding the immune system's ability to identify and attack cancer cells [81][82][83][84].Thus, they represent potential targets for immunomodulatory therapies in cancer.GAL3 and GAL9 are molecules that are overproduced by cancer cells and can suppress the immune response by binding to TCR and Tim-3, respectively, which are expressed on T cell lymphocytes (TCLs) [84,85].Hence, inhibiting or blocking GAL3 and GAL9 can help to enhance the immune response against cancer cells.This conclusion is supported by the preclinical studies which have shown that targeting GAL3 or GAL9 could inhibit tumor growth and improve the efficacy of the immune checkpoint inhibitors (ICIs) [84,86].Moreover, CD155 is a protein that is overexpressed by some cancer cells preventing the activation of immune cells [87].By blocking the CD155/TIGIT axis, the immune response can be enhanced and promote the elimination of cancer cells [88,89].Indeed, CD155 siRNAs can be effective in treating late-stage TNBC through immune escape [89].Interestingly, a previous study from our group showed that modulation of H 2 S has a potential impact on the immunosurveillance process [13].Our findings also have previously highlighted that dampening CBS and CSE expression levels using siRNAs consolidate the expression levels of MICA/B and ULBP2 in MDA-MB-231 cells, which in turn resulted in a marked increase in NK cells cytotoxicity upon co-culturing [90].Herein, the overexpression of miR-193a-3p and miR-548c-3p reduced the expression level of GAL3, GAL9, and CD155 and upregulated the expression levels of MICA and MICB in MDA-MD-231.These results provide the first evidence of the involvement of miR-193a-3p and miR-548c-3p in improving NK cells and TCLs immunosurveillance via modulating H 2 S production in BC.
While our results offer promising insights, several limitations warrant consideration.Our study predominantly focused on the MDA-MB-231 cell line, which may not fully capture the heterogeneity of BC in clinical populations.Further investigations encompassing diverse BC subtypes and clinical samples are needed to validate the translational potential of our findings.Additionally, elucidating the precise molecular mechanisms underpinning the pan-suppression of miR-193a-3p and miR-548c-3p on H 2 S synthesizing enzymes is essential.

Conclusion
In conclusion, our study presents compelling evidence that the three H 2 S synthesizing enzymes CBS, CSE, and 3MST are overexpressed in BC.Upon patient stratification based on 3MST expression level, 3MST was found to have higher transcript levels in BC patients at age < 40 years old, pre-menopausal, expressing high Ki-67 and having large tumor size (≥ 5 cm).Bioinformatics and insilico analysis predicted miR-193a-3p and miR-548c-3p as pan-suppressors of the three H 2 S synthesizing enzymes simultaneously.They were under-expressed in BC tissues.In-vitro study confirmed their pan-inhibition of the three H 2 S synthesizing in TNBC cells and attenuation of H 2 S production.Meanwhile, miR-193a-3p and miR-548c-3p suppressed the oncogenic profile of TNBCs and improved their immune response (Fig. 8).

Fig. 1 Fig. 2
Fig. 1 Screening of CBS, CSE, and 3MST in BC tissue.The expression profiles of the H 2 S-synthesizing enzymes, (A) CBS, (B) CSE, and (C) 3MST were analyzed in 25 BC patients using qRT-PCR and normalized to 18 S as an internal control.The three H 2 S-synthesizing enzymes showed a significant overexpression in BC tissues compared to their normal counterparts.Student t test was performed.**** = P < 0.0001 compared to normal counterparts

Fig. 8
Fig. 8 Pan-inhibition of the three H 2 S synthesizing enzymes by miRNA-193a and miRNA-548c modulates tumor cells and TME.Ectopic expression of miR-NA-193a and miRNA-548c suppress the expression of CBS, CSE, and 3MST enzymes decreasing the H 2 S production level and accordingly TNBC hallmarks such as cellular viability, cellular migration and colony forming ability have been significantly repressed.MiRNA-193a and miRNA-548c also modulate the immune-modulators such as GAL3/9, MICA/B, and CD155

Table 1
Characteristic features of BC female patients